title: Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.
abstract: Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Rowe
authors.forename: Kelly
authors.initials: K
authors.lastname: Combs
authors.forename: Sapna
authors.initials: S
authors.lastname: Deo
authors.forename: C
authors.initials: C
authors.lastname: Ensor
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
authors.forename: Xiaoge
authors.initials: X
authors.lastname: Qu
mesh_terms: D015121:6-Ketoprostaglandin F1 alpha / Q000032:analysis / Q000378:metabolism; D000331:Aequorin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000804:Angiotensin II / Q000032:analysis / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D006207:Half-Life; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D007118:Immunoassay; D007700:Kinetics; D009154:Mutation; D015202:Protein Engineering; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism
chemical_list: D007093:Imidazoles; D011719:Pyrazines; D000804:Angiotensin II; C017144:coelenterazine; D000331:Aequorin; D015121:6-Ketoprostaglandin F1 alpha